RESUMEN
BACKGROUND: The most aggressive form of breast cancer is triple-negative breast cancer (TNBC), which lacks expression of the estrogen receptor (ER) and progesterone receptor (PR), and does not have overexpression of the human epidermal growth factor receptor 2 (HER2). Treatment options for women with TNBC tumors are limited, unlike those with ER-positive tumors that can be treated with hormone therapy, or those with HER2-positive tumors that can be treated with anti-HER2 therapy. Therefore, we have sought to identify novel targeted therapies for TNBC. In this study, we investigated the potential of a novel phosphatase, NUDT5, as a potential therapeutic target for TNBC. METHODS: The mRNA expression levels of NUDT5 in breast cancers were investigated using TCGA and METABRIC (Curtis) datasets. NUDT5 ablation was achieved through siRNA targeting and NUDT5 inhibition with the small molecule inhibitor TH5427. Xenograft TNBC animal models were employed to assess the effect of NUDT5 inhibition on in vivo tumor growth. Proliferation, death, and DNA replication assays were conducted to investigate the cellular biological effects of NUDT5 loss or inhibition. The accumulation of 8-oxo-guanine (8-oxoG) and the induction of γH2AX after NUDT5 loss was determined by immunofluorescence staining. The impact of NUDT5 loss on replication fork was assessed by measuring DNA fiber length. RESULTS: In this study, we demonstrated the significant role of an overexpressed phosphatase, NUDT5, in regulating oxidative DNA damage in TNBCs. Our findings indicate that loss of NUDT5 results in suppressed growth of TNBC both in vitro and in vivo. This growth inhibition is not attributed to cell death, but rather to the suppression of proliferation. The loss or inhibition of NUDT5 led to an increase in the oxidative DNA lesion 8-oxoG, and triggered the DNA damage response in the nucleus. The interference with DNA replication ultimately inhibited proliferation. CONCLUSIONS: NUDT5 plays a crucial role in preventing oxidative DNA damage in TNBC cells. The loss or inhibition of NUDT5 significantly suppresses the growth of TNBCs. These biological and mechanistic studies provide the groundwork for future research and the potential development of NUDT5 inhibitors as a promising therapeutic approach for TNBC patients.
Asunto(s)
Pirofosfatasas , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Línea Celular Tumoral , Proliferación Celular , Pirofosfatasas/genética , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Estrogen receptor (ER)-negative cancers have a poor prognosis, and few targeted therapies are available for their treatment. Our previous analyses have identified potential kinase targets critical for the growth of ER-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple-negative" breast cancer (TNBC). Because phosphatases regulate the function of kinase signaling pathways, in this study, we investigated whether phosphatases are also differentially expressed in ER-negative compared to those in ER-positive breast cancers. We compared RNA expression in 98 human breast cancers (56 ER-positive and 42 ER-negative) to identify phosphatases differentially expressed in ER-negative compared to those in ER-positive breast cancers. We then examined the effects of one selected phosphatase, dual specificity phosphatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein microarray analyses of 172 proteins, including phosphoproteins. We identified 48 phosphatase genes are significantly differentially expressed in ER-negative compared to those in ER-positive breast tumors. We discovered that 31 phosphatases were more highly expressed, while 11 were underexpressed specifically in ER-negative breast cancers. The DUSP4 gene is underexpressed in ER-negative breast cancer and is deleted in approximately 50 % of breast cancers. Induced DUSP4 expression suppresses both in vitro and in vivo growths of breast cancer cells. Our studies show that induced DUSP4 expression blocks the cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, JNK1, RB, and NFkB p65 phosphorylation; and inhibits invasiveness of TNBC cells. These results suggest that that DUSP4 is a critical regulator of the growth and invasion of triple-negative breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Análisis por Matrices de Proteínas/métodos , Receptores de Estrógenos/metabolismo , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Ratones , Invasividad Neoplásica , Trasplante de Neoplasias , Fosforilación , Receptores de Estrógenos/deficiencia , Transducción de SeñalRESUMEN
PURPOSE: Rexinoids, agonists of nuclear retinoid X receptor (RXR), have been used for the treatment of cancers and are well tolerated in both animals and humans. However, the usefulness of rexinoids in treatment of breast cancer remains unknown. This study examines the efficacy of IRX4204, a highly specific rexinoid, in breast cancer cell lines and preclinical models to identify a biomarker for response and potential mechanism of action. EXPERIMENTAL DESIGN: IRX4204 effects on breast cancer cell growth and viability were determined using cell lines, syngeneic mouse models, and primary patient-derived xenograft (PDX) tumors. In vitro assays of cell cycle, apoptosis, senescence, and lipid metabolism were used to uncover a potential mechanism of action. Standard anti-HER2 therapies were screened in combination with IRX4204 on a panel of breast cancer cell lines to determine drug synergy. RESULTS: IRX4204 significantly inhibits the growth of HER2-positive breast cancer cell lines, including trastuzumab and lapatinib-resistant JIMT-1 and HCC1954. Treatment with IRX4204 reduced tumor growth rate in the MMTV-ErbB2 mouse and HER2-positive PDX model by 49% and 44%, respectively. Mechanistic studies revealed IRX4204 modulates lipid metabolism and induces senescence of HER2-positive cells. In addition, IRX4204 demonstrates additivity and synergy with HER2-targeted mAbs, tyrosine kinase inhibitors, and antibody-drug conjugates. CONCLUSIONS: These findings identify HER2 as a biomarker for IRX4204 treatment response and demonstrate a novel use of RXR agonists to synergize with current anti-HER2 therapies. Furthermore, our results suggest that RXR agonists can be useful for the treatment of anti-HER2 resistant and metastatic HER2-positive breast cancer.
Asunto(s)
Neoplasias de la Mama , Senescencia Celular , Receptor ErbB-2 , Animales , Femenino , Humanos , Ratones , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Retinoides/farmacología , Retinoides/uso terapéutico , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PREVENTION RELEVANCE: Our results show that everolimus delays mammary tumor formation in multiple mouse models, suggesting that mTOR inhibitors will be useful for the prevention of ER-negative and triple-negative breast cancer in humans. See related Spotlight, p. 787.
Asunto(s)
Neoplasias de la Mama , Neoplasias Mamarias Animales , Humanos , Ratones , Animales , Femenino , Receptores de Estrógenos/metabolismo , Serina-Treonina Quinasas TOR , Everolimus/farmacología , Everolimus/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/prevención & control , Neoplasias de la Mama/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and lacks effective targeted treatment strategies. Previously, we identified 33 transcription factors highly expressed in TNBC. Here, we focused on six sex determining region Y-related HMG-box (SOX) transcription factors (SOX4, 6, 8, 9, 10, and 11) highly expressed in TNBCs. Our siRNA screening assay demonstrated that SOX9 knockdown suppressed TNBC cell growth and invasion in vitro. Thus, we hypothesized that SOX9 is an important regulator of breast cancer survival and metastasis, and demonstrated that knockout of SOX9 reduced breast tumor growth and lung metastasis in vivo. In addition, we found that loss of SOX9 induced profound apoptosis, with only a slight impairment of G1 to S progression within the cell cycle, and that SOX9 directly regulates genes controlling apoptosis. On the basis of published CHIP-seq data, we demonstrated that SOX9 binds to the promoter of apoptosis-regulating genes (tnfrsf1b, fadd, tnfrsf10a, tnfrsf10b, and ripk1), and represses their expression. SOX9 knockdown upregulates these genes, consistent with the induction of apoptosis. Analysis of available CHIP-seq data showed that SOX9 binds to the promoters of several epithelial-mesenchymal transition (EMT)- and metastasis-regulating genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of genes involved in EMT (vim, cldn1, ctnnb1, and zeb1) and that SOX9 knockdown suppresses the expression of these genes. IMPLICATIONS: Our studies identified the SOX9 protein as a "master regulator" of breast cancer cell survival and metastasis, and provide preclinical rationale to develop SOX9 inhibitors for the treatment of women with metastatic triple-negative breast cancer.
Asunto(s)
Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Factor de Transcripción SOX9/genética , Neoplasias de la Mama Triple Negativas/patología , Regulación hacia Arriba , Animales , Apoptosis , Línea Celular Tumoral , Secuenciación de Inmunoprecipitación de Cromatina , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Células MCF-7 , Ratones , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.
Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Cromosomas Humanos Par 1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenocarcinoma del Pulmón/genética , Animales , Cromosomas Humanos Par 1/genética , Ensayo de Inmunoadsorción Enzimática , Aparato de Golgi/metabolismo , Humanos , Técnicas In Vitro , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Microtomografía por Rayos XRESUMEN
Estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple negative," breast cancer (TNBC) is a poor prognosis clinical subtype that occurs more frequently in younger women and is commonly treated with toxic chemotherapy. Effective targeted therapy for TNBC is urgently needed. Our previous studies have identified several kinases critical for TNBC growth. Since phosphatases regulate the function of kinase signaling pathways, we sought to identify critical growth-regulatory phosphatases that are expressed differentially in ER-negative, as compared to ER-positive, breast cancers. In this study, we examined the role of one of these differentially expressed phosphatases, the protein phosphatase Mg + 2/Mn + 2 dependent 1A (PPM1A) which is underexpressed in ER-negative breast cancer as compared to ER-positive breast cancers, in regulating TNBC growth. We found that PPM1A is deleted in ~40% of ER-negative breast cancers, and that induced expression of PPM1A suppresses in vitro and in vivo growth of TNBC cells. This study demonstrates that induction of PPM1A expression blocks the cell cycle and reduces CDK and Rb phosphorylation. These results suggest PPM1A is a crucial regulator of cell cycle progression in triple negative breast cancer. Our results also suggest that PPM1A loss should be explored as a predictive biomarker of CDK inhibitor sensitivity.
RESUMEN
PURPOSE: To test whether a novel rexinoid, LG100268, prevents the development of preinvasive and invasive estrogen receptor-negative mammary tumorigenesis in MMTV-erbB2 mice. EXPERIMENTAL DESIGN: For invasive breast cancer prevention, MMTV-erbB2 mice were treated with daily gastric gavage of vehicle, LG100268 (10 mg/kg), or LG100268 (100 mg/kg) for long term starting at 3 months of age. For preinvasive lesion study, mice were treated with daily gastric gavage of vehicle or LG100268 (100 mg/kg) for 4 months. RESULTS: Long-term treatment with LG100268 significantly prevented invasive mammary tumor development. Median time (age) to tumor development was delayed from 217 days in vehicle group to 357 days in low-dose group. In high-dose group, only 2 of 20 mice developed tumors after 430 days of treatment. Short-term treatment of LG100268 significantly prevented the development of preinvasive mammary lesions including hyperplasia and ductal carcinoma in situ. The cancer prevention effect was associated with reduced expression of Ki67 and cyclin D1 in mammary glands by >80%. CONCLUSION: Rexinoid LG100268 is an effective chemopreventive agent in preventing the development of both malignant and premalignant mammary lesions in MMTV-erbB2 mice.
Asunto(s)
Ácidos Nicotínicos/farmacología , Receptores de Estrógenos/metabolismo , Tetrahidronaftalenos/farmacología , Animales , Anticarcinógenos/uso terapéutico , Línea Celular Tumoral , Ciclina D1/biosíntesis , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/biosíntesis , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Transgénicos , Invasividad Neoplásica , Trasplante de Neoplasias , Receptor ErbB-2/genética , Factores de Tiempo , TransgenesRESUMEN
Tamoxifen reduces the relative risk of breast cancer developing from specific premalignant lesions. Many breast cancers that arise after tamoxifen treatment are estrogen receptor-alpha (ER-alpha)-negative, although premalignant lesions such as atypical ductal hyperplasia are highly ER-alpha-positive. The p53 null mouse mammary epithelial transplant model is characterized by ER-alpha-positive premalignant lesions that give rise to both ER-alpha-positive and ER-alpha-negative tumors. Given this progression from ER-alpha-positive to ER-alpha-negative lesions, we tested the ability of tamoxifen to block or delay mammary tumorigenesis in several versions of this model. In groups 1 and 2, p53 null normal mammary epithelial transplants were maintained in virgin mice. In groups 3 to 5, the p53 null and mammary transplants were maintained in mice continuously exposed to high levels of progesterone. In groups 6 and 7, transplants of the premalignant outgrowth line PN8a were maintained in virgin mice. Tamoxifen blocked estrogen signaling in these mice as evidenced by decreases in progesterone-induced lateral branching and epithelial proliferation in the mammary epithelium. Tamoxifen did not alter the elevated levels of progesterone in the blood while significantly reducing the circulating level of prolactin. Tamoxifen reduced tumor incidence in p53 null normal mammary epithelial transplants maintained in virgin mice from 55% to 5% and in progesterone-stimulated mice from 81% to 21%. The majority of the resultant tumors were ER-alpha-negative. Tamoxifen also significantly delayed tumorigenesis in the ER-alpha-positive high premalignant line PN8a from 100% to 75%. These results show that tamoxifen delays the emergence of ER-alpha-negative tumors if given early in premalignant progression.
Asunto(s)
Anticarcinógenos/farmacología , Receptor alfa de Estrógeno/deficiencia , Neoplasias Mamarias Experimentales/prevención & control , Tamoxifeno/farmacología , Animales , Procesos de Crecimiento Celular , Femenino , Neoplasias Mamarias Experimentales/sangre , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Progesterona/sangreRESUMEN
Despite the effectiveness of the selective estrogen receptor (ER) modulators in preventing ER-positive breast cancer, chemopreventive agents still need to be developed for the prevention of ER-negative breast cancers. The naturally occurring retinoids are promising agents for the prevention of human cancers but are too toxic for long-term chronic use. We previously demonstrated that the chemopreventive effects of the retinoids could be separated from the toxicity by using an RXR-selective retinoid, LGD1069. The studies described here demonstrate that LGD1069 effectively suppresses ER-negative tumor development in mouse mammary tumor virus-erbB2 transgenic mice with minimal toxicity. These studies suggest that receptor-selective retinoids are promising agents for the prevention of breast cancer and that they may be particularly useful in preventing ER-negative breast cancer.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias Mamarias Experimentales/prevención & control , Receptores de Ácido Retinoico/metabolismo , Tetrahidronaftalenos/farmacología , Factores de Transcripción/metabolismo , Animales , Bexaroteno , División Celular/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Genes erbB-2 , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Virus del Tumor Mamario del Ratón , Ratones , Ratones Transgénicos , Receptores de Estrógenos/fisiología , Receptores X Retinoide , Transgenes/efectos de los fármacos , Transgenes/genéticaRESUMEN
Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and women diagnosed with TNBC currently lack targeted treatment options. To identify novel targets for TNBC, we evaluated phosphatase expression in breast tumors and characterized their contributions to in vitro and in vivo growth of TNBC. Using Affymetrix microarray analysis of 102 breast cancers, we identified 146 phosphatases that were significantly differentially expressed in TNBC compared with estrogen receptor (ER)-positive tumors. Of these, 19 phosphatases were upregulated (0.66-fold; FDR = 0.05) in TNBC compared with ER-positive breast cancers. We knocked down 17 overexpressed phosphatases in four triple-negative and four ER-positive breast cancer lines using specific siRNAs and found that depletion of six of these phosphatases significantly reduced growth and anchorage-independent growth of TNBC cells to a greater extent than ER-positive cell lines. Further analysis of the phosphatase PTP4A3 (also known as PRL-3) demonstrated its requirement for G1-S cell-cycle progression in all breast cancer cells, but PTP4A3 regulated apoptosis selectively in TNBC cells. In addition, PTP4A3 inhibition reduced the growth of TNBC tumors in vivo Moreover, in silico analysis revealed the PTP4A3 gene to be amplified in 29% of basal-like breast cancers, and high expression of PTP4A3 could serve as an independent prognostic indicator for worse overall survival. Collectively, these studies define the importance of phosphatase overexpression in TNBC and lay the foundation for the development of new targeted therapies directed against phosphatases or their respective signaling pathways for TNBC patients. Cancer Res; 76(7); 1942-53. ©2016 AACR.
Asunto(s)
Proteínas de Neoplasias/genética , Monoéster Fosfórico Hidrolasas/genética , Proteínas Tirosina Fosfatasas/genética , Neoplasias de la Mama Triple Negativas/genética , Línea Celular Tumoral , Proliferación Celular , Humanos , Proteínas de Neoplasias/metabolismo , Pronóstico , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal , Análisis de Supervivencia , Neoplasias de la Mama Triple Negativas/mortalidadRESUMEN
Estrogen receptor-negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor-negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Proteínas Quinasas Asociadas a Muerte Celular/fisiología , Genes p53 , Mutación , Animales , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular , Proteínas Quinasas Asociadas a Muerte Celular/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Muerte Celular/genética , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapia , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Triple-negative breast cancers (TNBC) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs and 10 proved critical for anchorage-independent growth. In TNBC cells, an LPA-LPAR2-EZH2 NF-κB signaling cascade was essential for expression of interleukin (IL)-6, IL-8, and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for patients with TNBCs.
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Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Animales , Apoptosis/genética , Apoptosis/inmunología , Línea Celular Tumoral , Supervivencia Celular/inmunología , Citocinas , Femenino , Xenoinjertos , Humanos , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-8/antagonistas & inhibidores , Interleucina-8/genética , Ratones , Ratones Desnudos , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/patología , Modelos de Riesgos Proporcionales , Transducción de Señal , Transfección , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
In pursuit of effective therapeutic agents for the estrogen receptor (ER)-negative breast cancer, we previously showed that bexarotene reduced mammary tumor development by 75% in ErbB2 mice. To further improve the effectiveness of breast cancer prevention, we have now investigated the effects of a combinatorial therapy consisting of two cancer preventive drugs. On the basis of the hypothesis, rexinoid LG100268 plus tamoxifen would more effectively prevent the development of both ER-positive and ER-negative breast cancer. We treated p53-null mammary gland mice with tamoxifen and LG100268, individually and in combination. By 60 weeks of age, vehicle-treated mice developed tumors in 52% of transplanted mammary glands, whereas mice treated with tamoxifen and LG100268 developed tumors in only 13% of transplanted mammary glands. To further define the mechanistic effects of this combinatorial treatment, we investigated the effects of tamoxifen and LG100268 on mammary tissue biomarkers. In mammary tissue harvested before tumor development, the proliferation markers Ki67 and cyclin D1 were significantly reduced in mice treated with the combination therapy. In addition, the rexinoid target genes ABCA1 and ABCG1 were induced in both the rexinoid and combination treatment groups, whereas expression remained constant in tamoxifen group. These results show that tamoxifen-LG100268 combinatorial treatment is more effective in preventing mammary tumors than either agent alone. In addition, these studies have identified relevant tissue biomarkers that can be used to show the effect of these agents on mammary tissue. These results support the development of clinical trials of antiestrogen and rexinoid combinatorial therapy for the prevention of patients with high-risk breast cancer.
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Conservadores de la Densidad Ósea/farmacología , Neoplasias Mamarias Experimentales/prevención & control , Ácidos Nicotínicos/farmacología , Receptores de Estrógenos/metabolismo , Tamoxifeno/farmacología , Tetrahidronaftalenos/farmacología , Proteína p53 Supresora de Tumor/fisiología , Animales , Western Blotting , Femenino , Técnicas para Inmunoenzimas , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The chemopreventive effects of three agents, rexinoid bexarotene, tyrosine kinase inhibitor gefitinib, and celecoxib, were tested on mammary tumor development arising in p53-null mammary epithelium. The rexinoid bexarotene was the most efficacious inhibitor as it reduced mammary tumor development by 75% in virgin mice and significantly delayed mean tumor development by 98 days in hormone-stimulated mice. The tyrosine kinase inhibitor gefitinib reduced mammary tumor incidence by 50% in virgin mice but did not significantly delay mean tumor latency in hormone-stimulated mice. Celecoxib did not reduce tumor incidence or mean tumor latency in either of the two models. The high doses of the rexinoid and the tyrosine kinase inhibitor did not affect the progression of tumors arising from the premalignant mammary outgrowth line, PN8a. A comparison of these agents with tamoxifen shows the superiority of tamoxifen in preventing tumor development in p53-null mammary cells. Similarly, a comparison of the results of the p53 model with other transgenic models in their response to the chemopreventive agents showed that mammary tumors arising from different oncogenic events will respond differently to the different agents.
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Modelos Animales de Enfermedad , Glándulas Mamarias Animales/efectos de los fármacos , Neoplasias Mamarias Experimentales/prevención & control , Pirazoles/uso terapéutico , Quinazolinas/uso terapéutico , Sulfonamidas/uso terapéutico , Tetrahidronaftalenos/uso terapéutico , Proteína p53 Supresora de Tumor/fisiología , Animales , Anticarcinógenos/uso terapéutico , Bexaroteno , Celecoxib , Inhibidores de la Ciclooxigenasa/uso terapéutico , Receptores ErbB/antagonistas & inhibidores , Femenino , Gefitinib , Técnicas para Inmunoenzimas , Incidencia , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones NoqueadosRESUMEN
Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)-negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor-induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/análisis , Receptores ErbB/antagonistas & inhibidores , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/prevención & control , Lesiones Precancerosas/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Receptor ErbB-2/análisis , Animales , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma Intraductal no Infiltrante/tratamiento farmacológico , Ciclina D1/efectos de los fármacos , Ciclina D1/metabolismo , Factor de Crecimiento Epidérmico/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Epirregulina , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lapatinib , Neoplasias Mamarias Experimentales/química , Ratones , Ratones Transgénicos , Lesiones Precancerosas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Estrógenos/análisis , Transducción de Señal/efectos de los fármacosRESUMEN
Genetically engineered mouse cancer models are among the most useful tools for testing the in vivo effectiveness of the various chemopreventive approaches. The p53-null mouse model of mammary carcinogenesis was previously characterized by us at the cellular, molecular, and pathologic levels. In a companion article, Medina et al. analyzed the efficacy of bexarotene, gefitinib, and celecoxib as chemopreventive agents in the same model. Here we report the global gene expression effects on mammary epithelium of such compounds, analyzing the data in light of their effectiveness as chemopreventive agents. SAGE was used to profile the transcriptome of p53-null mammary epithelium obtained from mice treated with each compound versus controls. This information was also compared with SAGE data from p53-null mouse mammary tumors. Gene expression changes induced by the chemopreventive treatments revealed a common core of 87 affected genes across treatments (P < 0.05). The effective compounds, bexarotene and gefitinib, may exert their chemopreventive activity, at least in part, by affecting a set of 34 genes related to specific cellular pathways. The gene expression signature revealed various genes previously described to be associated with breast cancer, such as the activator protein-1 complex member Fos-like antigen 2 (Fosl2), early growth response 1 (Egr1), gelsolin (Gsn), and tumor protein translationally controlled 1 (Tpt1), among others. The concerted modulation of many of these transcripts before malignant transformation seems to be conducive to predominantly decrease cell proliferation. This study has revealed candidate key pathways that can be experimentally tested in the same model system and may constitute novel targets for future translational research.
Asunto(s)
Biomarcadores de Tumor/genética , Modelos Animales de Enfermedad , Neoplasias Mamarias Experimentales/genética , Lesiones Precancerosas/genética , Pirazoles/uso terapéutico , Quinazolinas/uso terapéutico , Sulfonamidas/uso terapéutico , Tetrahidronaftalenos/uso terapéutico , Proteína p53 Supresora de Tumor/fisiología , Animales , Anticarcinógenos/uso terapéutico , Bexaroteno , Biomarcadores de Tumor/metabolismo , Celecoxib , Inhibidores de la Ciclooxigenasa/uso terapéutico , Receptores ErbB/antagonistas & inhibidores , Femenino , Gefitinib , Perfilación de la Expresión Génica , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Lesiones Precancerosas/tratamiento farmacológico , Lesiones Precancerosas/patología , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
BACKGROUND: The rexinoid bexarotene (LGD1069, Targretin) is a highly selective retinoid x receptor (RXR) agonist that inhibits the growth of pre-malignant and malignant breast cells. Bexarotene was shown to suppress the development of breast cancer in transgenic mice models without side effects. The chemopreventive effects of bexarotene are due to transcriptional modulation of cell proliferation, differentiation and apoptosis. Our goal in the present study was to obtain a profile of the genes modulated by bexarotene on mammary gland from three transgenic mouse mammary cancer models in an effort to elucidate its molecular mechanism of action and for the identification of biomarkers of effectiveness. METHODS: Serial analysis of gene expression (SAGE) was employed to profile the transcriptome of p53-null, MMTV-ErbB2, and C3(1)-SV40 mammary cells obtained from mice treated with bexarotene and their corresponding controls. RESULTS: This resulted in a dataset of approximately 360,000 transcript tags representing over 20,000 mRNAs from a total of 6 different SAGE libraries. Analysis of gene expression changes induced by bexarotene in mammary gland revealed that 89 genes were dysregulated among the three transgenic mouse mammary models. From these, 9 genes were common to the three models studied. CONCLUSION: Analysis of the indicated core of transcripts and protein-protein interactions of this commonly modulated genes indicate two functional modules significantly affected by rexinoid bexarotene related to protein biosynthesis and bioenergetics signatures, in addition to the targeting of cancer-causing genes related with cell proliferation, differentiation and apoptosis.
RESUMEN
The oncogene erbB2 is overexpressed in 20% to 30% human breast cancers and is most commonly overexpressed in estrogen receptor (ER)-negative breast cancers. Transgenic mice expressing erbB2 develop ER-negative mammary tumors, mimicking human breast carcinogenesis. Previously, we have shown that activator protein 1 (AP-1) regulates proliferation of ER-negative breast cancer cells. We hypothesized that blockade of AP-1 in mouse mammary epithelial cells will suppress ER-negative tumorigenesis induced by erbB2. Trigenic erbB2 mice were generated by crossing a bigenic pUHD-Tam67/MMTV-rtTA mouse to a MMTV-erbB2 mouse. The resulting trigenic mice develop tumors and express a doxycycline-inducible c-Jun dominant negative mutant (Tam67) in the mammary glands. In vivo AP-1 blockade by Tam67 expression started delayed mammary tumor formation in MMTV-erbB2 mice by more than 11 weeks. By 52 weeks of age, 100% (18 of 18) of the untreated animals had developed mammary tumors, whereas 56% (9 of 16) of the doxycycline-treated trigenic mice developed tumors. In addition, the tumors that arose in the AP-1-blocked erbB2 mice failed to express Tam67. Twenty-five percent of the doxycycline-treated MMTV-erbB2 mice survived more than 72 weeks of age without developing mammary tumors. Examination of normal-appearing mammary glands from these mice showed that AP-1 blockade by Tam67 also significantly prevents the development of premalignant lesions in these glands. The expression of erbB2 either in normal mammary tissue or in mammary tumors was not altered. Our results show that blocking the AP-1 signaling in mammary cells suppresses erbB2-induced transformation, and show that the AP-1 transcription factor is a critical transducer of erbB2. These results provide a scientific rationale to develop targeted drugs that inhibit AP-1 to prevent the development of ER-negative breast cancer.